Effect of MAPK/NF-κB signaling pathway on extracellular release of HMGB1induced by hypoxia in laryngeal Hep-2carcinoma cells
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摘要: 目的:观察缺氧对喉癌细胞高迁移率族蛋白B1(HMGB1)释放的诱导作用,并探讨其可能的机制。目的:采用人喉癌细胞株Hep-2,观察缺氧(1% O2)培养不同时间对细胞培养上清液中HMGB1含量、细胞HMGB1蛋白和mRNA的影响,及不同浓度的MAPK信号通路抑制剂(PD98059、SP600125、SB202190)、NF-κB抑制剂(PDTC)对缺氧诱导的HMGB1释放的影响。上清液HMGB1含量和细胞HMGB1蛋白表达水平分别采用酶联免疫吸附试验和Western blot检测;细胞HMGB1 mRNA表达水平采用实时荧光定量PCR法检测。结果:培养上清液中HMGB1含量和细胞HMGB1蛋白表达水平均于缺氧诱导12 h后升高,呈时间依赖性;细胞HMGB1 mRNA表达水平于缺氧诱导6 h开始升高,并随诱导时间延长而进一步升高;20 μmol/L PD98059、SP600125和50 mg/L PDTC对缺氧诱导的HMGB1释放有不完全的抑制作用。结论:缺氧诱导喉癌细胞释放HMGB1,其机制可能与MAPK/NF-κB信号通路有关。Abstract: Objective: To investigate the extracellular release of high mobility group box 1(HMGB1) in laryngeal Hep-2 carcinoma cells induced by hypoxia and its possible mechanism.Method: The changes of HMGB1 concentration in the culture medium as well as HMGB1 protein and mRNA expression in Hep-2 cells were investigated after the cells were cultured with 1% O2 for different durations. Inhibitory effects of MAPK pathway inhibitors(PD98059, SP600125, and SB202190) and nuclear NF-κB pathway inhibitor(PDTC) with various concentrations on extracellular HMGB1 release were observed in hypoxia-induced Hep-2 cells. The HMGB1 concentration and HMGB1 protein expression were measured by enzyme-linked immunosorbent assay(ELISA) and western blot, respectively. The HMGB1 mRNA expression was determined by real-time quantitative PCR(RT-PCR).Result: The HMGB1 concentration in the culture medium and the HMGB1 protein expression in Hep-2 cells increased after the cells were subjected to hypoxia culture for 12 h in a time-dependent manner. The level of HMGB1 mRNA expression in Hep-2 cells increased after the cells were induced by hypoxia for 6h PD98059 and SP600125 with 20 μmol/L and PDTC with 50 mg/L partly inhibited extracellular release of HMGB1 in hypoxia-cultured Hep-2 cells.Conclusion: Hypoxia induces laryngeal carcinoma cells to release HMGB1, which may be related to MAPK/NF-κB signaling pathway.
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